Qualitative requirements of essential fatty acids for juvenile Penaeus monodon

Aquacwltllre ELSEVIER

Aquaculture 147 ( 1996) 275-29 1

Qualitative requirements of essential fatty acids for juvenile Penaeus monodon Zuridah 0. Merican Department

*,

K.F. Shim

of Zoology, National University of Singapore, Kent Ridge, 119260, Singapore

Accepted 3 I May 1996

Abstract The requirements of essential n - 3 and n - 6 fatty acids (EFA) for juvenile Penaeus were assessed qualitatively in terms of weight and survival responses by feeding diets excluding each fatty acid separately. Purified diets contained 5% lipid mixture of 16:O and 18: 1n - 9 (conditioning/control diet), 5% cod liver oil (reference diet) or 4.2% lipid mixture of 16:0 and 18: 1n - 9 and 0.8% mixture of four fatty acids of the five EFAs; 18:2n - 6, 18:3n - 3, 20:4n - 6, 20:5n - 3 and 22:6n - 3. In a separate experiment, shrimp were fed diets with I% 20:5n - 3, 1% 22:6n - 3 or 1% mixture of 20:5n - 3 and 22:6n - 3 added to a 4% mixture of 16:O and 18: 1n - 9 as dietary lipid. In both experiments, feeding the reference diet with 5% cod liver oil resulted in higher growth than diets supplemented with pure polyunsaturated fatty acids (PUFAs). In Experiment 1, weight gain of shrimp fed diets without supplements of 18:2 rl - 6, 20:4n - 6 and 20:5n - 3 ranged from 275 to 301%. A requirement for pre-formed 18:3n - 3 and 22:6n - 3 was indicated by the significant declines (P < 0.05) in weight gain (189 and 195%, respectively) when shrimp were fed diets without these fatty acids. When fed the diet supplemented with only 1% 22:6n - 3 as dietary EFA, weight gain of shrimp (359%) was significantly (P < 0.05) higher than of shrimp fed the diet supplemented with 1% 20:5n - 3. However, the addition of 22:6n - 3 to 20:5n - 3 significantly (P < 0.05) improved growth. Survival responses were not related to qualitative differences in n - 3 and n - 6 PUFAs supplemented into diets. Tissue levels of saturates and monounsaturates decreased with the supplementation of n - 3 and n - 6 PUFAs. No de novo synthesis of 18:3n - 3 was evident. The chain elongation and desaturation of 20:5n - 3 to 22:6n - 3 and the retroconversion of 22:6n - 3 to 20:5n - 3 occurred. These results showed differences in the mono&n

* Corresponding author. Tel.: 65 7722708; fax: 65 7792486; e-mail: scip3008@nus,sg. 0044-8486/96/$15.00 Copyright 0 1996 Elsevier Science B.V. All rights reserved. PII SOO44-8486(96)0 1379-8

276

qualitative requirements other penaeids.

2.0. Merican, K.F. Shim /Aquaculture

of EFA and bioconversion

147 (1996) 275-291

capabilities

of P. monodon

as compared with

Keywords: Penaeus monodon; Feeding and nutrition - crustacean; Fats and fatty compounds; Growth -

crustacean

1. Introduction Linoleic (18:2n - 6), linolenic (18:3n - 31, arachidonic (20:4n - 6), eicosapentaenoic (20:5n - 3) and docosahexaenoic (22:6n - 3) acids have been determined as essential for penaeid nutrition and physiology and must be supplied in the diets of several penaeid species (P. juponicus, Kanazawa et al., 1979a; P. indicus, Read, 1981; P. chinensis, Xu et al., 1994). There are no specific studies to determine the essential fatty acids for P. monodon, but by feeding marine oils, Wang (1988) attributed the good growth response to the presence of n - 3 highly unsaturated fatty acids (HUFAS). The feeding of this species has been largely based on formulations originally designed for P. juponicus. Due to known differences among shrimp species such as feeding habitats and climatic conditions, the requirements for essential fatty acids (EFAs) may differ. Chen (1995) postulated that the requirements of P. monodon for several nutrients may be closer to fish rather than other penaeids. Whether this applies to lipids is not known. The nutritional requirements of EFAs may be affected by the biosynthetic and bioconversion abilities of the shrimp. Dietary 18:2n - 6 and 18:3n - 3 were found to be required since de novo synthesis was absent in P. juponicus (Kanazawa et al., 1979b) and in P. setiferus (Bottino et al., 1980). The capacity to chain elongate and desaturate 18:3n - 3 and 18:2n - 6 to the C20 and C22 HUFA also varies among species (Kanazawa et al., 1979b; Bottino et al., 1980; Kayama et al., 1980; Read, 1981; Araujo and Lawrence, 1991; Xu et al., 1994). The relative efficacy of n - 3 HUFAs was also affected by the bioconversion abilities and requirements in P. juponicus (Teshima et al., 1992) and in marine fish (Takeuchi et al., 1990, 1992). Xu et al. (1994) showed that the essential fatty acid values in the diet for P. chinensis were 18:2n - 6 < 18:3n - 3 < 20:4n - 6 < 22:6n - 3. In the earlier studies on EFA requirements, the conventional methodology used was to evaluate the effects of pure fatty acids added as a single EFA source in a purified diet or to study the effects of fatty acids present as triacylglycerols. With the former methodology, the associative effects of a combination of EFAs in diets, which may affect biosynthetic capabilities and requirements, are not known. Whilst with triacylglycerols, it was not possible to identify the effects of the individual EFA. Hence, the objective of this study was to assess the nutritive value of each EFA in the presence of the remaining n - 3 and n - 6 PUFAs. Weight gain and survival as well as tissue composition were used to evaluate responses to the deletion of each EFA separately in a purified diet. In a separate experiment, the efficacy of 20:5n - 3 and 22:6n - 3 were determined in shrimp fed diets containing these HUFAs as a single EFA source.

2.0. Merican, K.F. Shim/Aquaculture

2. Materials

147 (1996) 275-291

277

and methods

2.1. Experimental

tank system

All diet evaluations were conducted using a semi-closed recirculating system comprising 100-l (0.29 m2 bottom) plastic tanks. The flow rate to each tank was maintained at 75 1 h-r throughout the experimental period of 35 days. Temperature, pH and salinity averaged 28.5 + 0.7”C, 7.83 f 0.12 and 25.13 f 1.72%~ respectively. Mean values of total NH,-N, NO,-N and NO,-N were 0.05, 2.12 and 0.03 mg I-‘, respectively. 2.2. Experimental

shrimp

Postlarvae Penaeus monodon purchased from commercial hatcheries at PL ,0 _ ,5 were reared to size in outdoor concrete tanks. Juvenile shrimp of approximate 0.05 to 0.1 g were selected by size and stocked into experimental tanks at a density of 20 shrimp per tank. All shrimp were fed a conditioning diet containing a mixture of 16:0 and 18: 1 n - 9 as dietary lipid for 7 days, to reduce EFA reserves in tissues, before the trials with fatty acid treatment diets were initiated. After preconditioning, a random subsample of 30 shrimp were collected. Their weight divided by the number of shrimp gave the initial wet weight. Five replicates were recorded. The mean of these weights were assumed equal in all experimental tanks for each trial. This estimated wet weight rather than individual weights was used because considerable stress occurred from handling during the weighing process. 2.3. Diets A basal purified diet was formulated to contain 47% protein. Lipid extracted fish soluble concentrate and squid meal were used as attractants. The chemical composition of the diets used in Experiments 1 and 2 are given in Table 1. The lipid mixture comprised 5% fatty acid, 0.7% choline chloride and 0.3% vitamin A acetate as antioxidant (Table 2). The conditioning/control diet contained 5% of a mixture of 16:0 and 18: 1 n - 9, whilst the five treatment diets in Experiment 1 contained 4.2% of this mixture and 0.8% mixture of four out of five EFAs to be tested. Thus in Diet LI, 18:2n - 6 was deleted from the lipid mixture and in Diets NC, AR, EP and DH, 18:3n - 3, 20:4n - 6, 20:5n - 3 and 22:6n - 3, respectively, were deleted. In Experiment 2, diets contained a 4% mixture of 16:O and 18:l n - 9 and either 1% 20:5n - 3 (Diet ES), 22:6n - 3 (Diet DS) or a mixture of equal proportions of these HUFAs as dietary lipid (Diet S). The purity of fatty acids used ranged from > 99% for 16:0, 18:ln - 9, 18:2n - 6, 18:3n - 3 and 20:4n - 6, > 95% for 22:6n - 3 and > 50% for 20:5n - 3. The fatty acid composition of diets are given in Table 3. The basal purified diet contained 0.4% 18:2n - 6 and small proportions of 16:0, 18:0, 18: 1 n - 9 and 18:3n - 3 fatty acids, which was reflected in all diets. The composition of EFAs was consistent with the formulation in Table 2, except for the content of 18:2n - 6 which was present at 0.4% even in Diet LI from which this fatty acid was deleted. Cod liver oil, used as dietary lipid for the reference diet, also contained 4% of total fatty acids of 18:2n - 6. This contributed to the high level of this fatty acid in the reference diet.

278 Table 1 General composition

2.0. M&can,

and proximate

K.F. Shim/Aquaculture

analysis

147 (1996) 275-291

of purified diet g per 100 g (dry diet)

Ingredients Casein (vitamin free) Gelatin Cornstarch Fish soluble concentrate Squid meal b Vitamin mixture ’ Mineral mixture d

a

K,HW Lecithin ’ Cholesterol ’ Sodium alginate Sodium hexametaphosphate Alpha cellulose Vitamin C 2-6 Di-tert-butyl-4methylphenol Lipid mixture Choline chloride Vitamin E acetate(1360 IU g-l) Lipid g Total Proximate analysis of diet C% dry matter) Crude protein Total lipid Ash Gross energy (kcal g- ’ )

33.0 14.0 20.0 1.0 1.0 4.0 7.0 2.0 2.0 0.5 2.5 1.5 5.2 0.2 0.1 0.7 0.3 5.0 loo.0

42.89f3.13 7.43 + 0.39 10.08 + 0.65 5.93kO.15

a Lipid extracted, Sopropfiche, France. b Lipid extracted, produced in Malaysia. ’ Vitamin mix (mg kg- ’ of dry diet): thiamin HCl, 200; riboflavin, 320; pyridoxine HCI, 120; dl-Ca pantothenate, 600; nicotinic acid, 1000; d-biotin, 40; folic acid, 200; cyanocobalamin, 40; inositol, 4000, vitamin A acetate, 1000; cholecalciferol, 40; menadione, 40; para amino benzoic acid, 1200; vitamin C, 2000; vitamin E dl-cu-tocopherol, 2000; sucrose, 27 200. d AIN mineral mix, ICN Biochemicals, USA. e 98% phospholipids, Lucas-Meyer, Gmbh. ’ 98% pure, Sigma Chemicals. g Pure fatty acids.

All diet ingredients were ground to pass through Diet ingredients were mixed using a kitchen mixer orifice die. Pellet strands were freeze dried. Pellets type grinder (Bra& and sifted through 250-500~pm of particle size. Diets were kept in sealed plastic bags - 40°C. In both experiments, shrimp were also fed the together with the treatment diets. The control diet

a 500~pm sieve prior to mixing. and cold extruded with a l-mm were crumbled in a hammer mill sieves to obtain the specific range flushed with nitrogen and stored at control diet and a reference was used to obtain growth

diet and

2.0. Merican,

K.F.

Shim/AquacuLure

Table 2 Percent ourified fattv acids added to diets in Exoeriments Lipid

I47

(19961275-291

279

1 and 2 (% of diet)

Diets Experiment

Experiment

Ref.

cont. 16:O 18:ln-9 18:2n-6 18:3n-3 20:4n - 6 205n - 3 22:6n - 3 Cod liver oil Total

1

2.5 2.5

5.0 5.0

2

LI

NC

AR

EP

DH

ES

DS

S

2.1 2.1

2.1 2.1 0.2

2.1 2.1 0.2 0.2

2.1 2.1 0.2 0.2 0.2

2.1 2.1 0.2 0.2 0.2 0.2

2.0 2.0

2.0 2.0

2.0 2.0

1.0

0.5 0.5

5.0

5.0

0.2 0.2 0.2 0.2

0.2 0.2 0.2

0.2 0.2

0.2

5.0

5.0

5.0

5.0

1.0

5.0

5.0

survival responses and fatty acid composition of tissues as baseline information in the absence of supplemented EFA. The reference diet provided growth and survival responses and fatty acid composition of tissues of shrimp fed a diet with a complete EFA profile. This diet was also used to verify that adequate experimental conditions and uniform quality of juveniles were maintained for all trials.

Table 3 Fatty acid composition determinations Fatty acid

of diets for Experiments

Diets Experiment

14:o 16:O 16:ln-7 18:O 18:ln-9 18:2n-6 18:3n -3 18:4n-3 20:o 20:ln-9 20:4n - 6 20:5n - 3 22:o 22:ln-9 22:6n - 3 Totals Fatty acids n-3/n-6

I and 2 (% dry matter) a. Data are mean values of three

1

Basal

Control

0.20

2.39

0.05 0.06 0.39 0.05

2.38 0.36 0.04

0.75

a n - 3 polyunsaturates:

5.17

Experiment Ref. 0.09 0.72 0.20 0.10 0.72 0.58 0.14 0.08 0.02 0.12 0.03 0.45 0.03 0.08 0.25 3.61

1.50 18:3n - 3, 205

2

LI

NC

AR

EP

DH

ES

DS

S

2.17

2.00

2.05

I .98

2.37

2.13

0.05 I .95 0.38 0.2 1

0.05 1.80 0.5 1 0.05

0.04 0.46 0.21

0.05 2.05 0.54 0.22

0.05 1.81 0.59 0.26

2.08 0.01 0.05 1.69 0.40 0.06

2.20 0.01 0.06 I .83 0.41 0.06

0.01 0.19 0.22

0.19 0.2 I

0.18 0.21

0.2 1 0.18

0.82

0.18

0.22

0.22

0.22

5.36 1.07

5.03 0.6 1

5.11 1.39

5.24 0.61

1.92

5.47 0.55

- 3, 22:6n - 3. n - 6 polyunsaturates:

5.11 2.20

0.06 1.93 0.39 0.06

0.45

1.03

0.60

5.60 2.79

5.62 2.70

18:2n - 6, 20:4n

- 6.

280

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147 (1996) 275-291

2.4. Feeding and monitoring Five tanks were assigned randomly for each treatment, control and reference diet. Feeding commenced after the conditioning period. Shrimp were fed the experimental diets for 28 days at approximately 20% body weight day-‘, which was equally divided among five daily feedings at 800, 12:00, 15:00, 17:00 and 24:O0. Tanks were siphoned clean of waste feed daily. Before feeding the treatment diets, the number of shrimp per tank was adjusted to 20 shrimp. Dead shrimp were removed daily and the survival was recorded daily. 2.5. Final weights and tissue sampling On termination of the growth trials, shrimp were starved for 24 h prior to weighing and tissue sampling. Hepatopancreas and tail muscle from shrimp from each tank were separated and pooled as single samples. Samples were kept at - 40°C for further analysis. 2.6. Chemical analyses Total lipids were extracted from tissues and diets (Folch et al., 1957). Tail muscle lipids were separated into polar and neutral lipids (Juaneda and Rocquelin, 1985). Fatty acid methyl esters were derivatised with boron trifluoride (AOAC, 1980) from polar and neutral lipids of tail muscle and total lipids of hepatopancreas and diets. Internal standards, 17:0, heptadecanoic methyl ester (Sigma, USA) and 23:0 tricosanoic methyl ester (Nu-chek, USA) were used to quantify the fatty acids (Joseph and A&man, 1992). A Shimadzu 9A gas chromatograph with a Thermon 3000A capillary column (25 m L X 0.25 mm ID) was used. 2.7. Statistical analysis The mean percent weight gain and composition of tissues were analysed by analysis of variance (SAS, 1987). Percent survival data were subjected to arcsine transformation. Means were compared using Duncan’s multiple range test to determine significant (P < 0.05) differences between individual treatments.

3. Results 3.1. Growth and survival In Experiment 1, significant (P < 0.05) differences in weight gain and survival of shrimp fed the various diets (Fig. 1) were observed. The highest weight gain (464%)

Fig. 1. The effect of the deletion of n - 3 and n - 6 PUFA on growth (A) and survival (B) of juvenile P. monodon fed the experimental diets and the effect of feeding experimental diets containing 1% 20:5n - 3, 1% 22:6n -3 and their combination on growth (C) and survival (D) on juvenile P. monodon. Values are mean f SD. Means with the same letters are not significantly (P < 0.05) different.

Z.O. Merican,

282

K.F. Shim/Aquaculture

147 (1996) 275-291

was shown by shrimp fed the purified diet containing cod liver oil and the lowest (151%) in shrimp fed the control diet. Growth was significantly (P < 0.05) reduced when either 18:3n - 3 (Diet NC) or 22:6n - 3 (Diet DH) was not provided in the diet. Weight gain was 195 and 189%, respectively, and it was not significantly (P > 0.05) different from that of shrimp fed the control diets. No significant (P > 0.05) differences in growth occurred in shrimp fed Diets EP, AR and LI. The supplementation of EFAs improved survival. Survival was not significantly (P > 0.05) related to the qualitative differences in composition of diets. In Experiment 2, percent weight gain of shrimp fed diets with 1% 22:6n - 3 (Diet DS> as dietary EFA was significantly (P < 0.05) higher than when the dietary EFA was substituted with 20:5n - 3 (Diet ES). Intermediate weight gains were obtained with shrimp fed a combination of 20:5n - 3 and 22:6n - 3. The type and composition of

Table 4 Fatty acid content (mg g- ’ dry matter) in hepatopancreas, to (initial) and after conditioning Fatty acid

Hepatopancreas

polar and neutral lipid of tail muscle of shrimp prior

Tail muscle Polar lipids

140 16:0 16:ln-7 18:O 18:ln-9 18:2n-6 18:3n-3 20: 1n - 9 20:4n-6 20:5n-3 22:o 22:6n-3 Sat a Monounsat. b n-3 PUFA ’ n-6 PUFA d Total n-3/n-6 PUFA ’

Neutral lipids

Initial

After

% Change

Initial

After

% Change

Initial

After

4Sa 38.la 5.7a 10.9a 19.8 a 18.6a 2.la 5.9a 6.6a 9.2a 2.8a 16.6a 56.4a 31.4a

0.4b 15.7b 2.3b 3.9b 19.6a 5.8b 1.2b 0.4b 3.1b 5.5b 0.3b 4.3b 20.5b 22.6b

91.2 58.9 60.4 64.2 0.9 68.7 42.6 92.7 53.6 40.9 88.5 73.9 63.7 28.2

0.2a 4.0a 0.3a 1.4a 1.3b 1.7a 0.2a nd 1.Oa 2.4a 0.2a 2.9a 5.6a 1.6b

O.lb 3.5b 0.2a 0.9b 2.7a 1.4b O.lb nd 0.5b 1.Ob O.lb 1.5b 4.8b 3.0a

33.3 11.8 27.3 38.0 t 110.0 23.3 52.6

tr

49.5 58.9 63.2 47.6 14.8 + 84.0

0.1 0.6a O.la 0.3a 0.4b 0.5a O.la O.la 0.3a 0.5a 0.1 0.6a 1.Oa 0.6b

0.4a O.la 0.3a 0.8a 0.5a O.la O.la 0.2a 0.3b tr 0.5b 0.9a 1.Oa

+ 11.1 t 86.4 3.6 11.1 28.6 20.0 39.6 62.5 20.6 9.5 t 59.0

27.9a

9.5b

66.2

5.5a

2.6b

52.8

1.3a

0.9b

30.5

25.2a

8.9b

64.6

2.7a

1.8b

32.7

0.8a

0.7a

16.1

61.9b 1.1

56.2

15.6a 2.1

12.2b 1.4

21.5

3.7a 1.6

3.4a 1.3

6.9

141.4a 1.1

% Change 20.0 5.2

a Saturated: 14:0, 16:0, 18:0. (20x0 not detected.) b Monounsaturates: 16:ln-7, 18:ln-9, 20:ln-9. ’ n - 3 polyunsaturated: 18:3n - 3, 20:5n - 3, 22:6n - 3. d n - 6 polyunsaturated: 18:2n - 6, 20:4n - 6. Data are means of five groups of shrimp (n = 100). Means within same rows with the same letters are not significantly (P > 0.05) different. nd, not detected; tr, trace ( < 0.05 mg g-l>.

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I47 11996) 275-291

283

pure HUFAs supplemented into diets did not significantly (P > 0.05) affect survival responses. Without any HUFA, survival of shrimp was reduced to 52%. 3.2. Tissue fatly acid composition Table 4 shows the changes in tissue fatty acid composition of preconditioned shrimp. The total fatty acid content decreased from a mean of 141.4 to 61.9 mg g- ’ in the hepatopancreas. Smaller changes were observed in the polar and neutral lipids of tail muscle (21.5 and 6.9%, respectively). Dietary 16:0 was preferentially catabolised and 18: 1n - 9 relative to 16:O was preferentially incorporated into both the hepatopancreas and polar lipids of tail muscle. Long chain PUPAS decreased significantly (P < 0.05). Endogenous 22:6n - 3 decreased by 73.9% in the hepatopancreas, but only 47.6% in the polar lipids of tail muscle. In contrast, changes in the content of 18:3n - 3 and 20:5n - 3 were higher in polar lipids as compared with the hepatopancreas. Minor changes were observed for 18:2n - 6 and 18:3n - 3 in neutral lipids. Tissue fatty acid content of shrimp fed control, reference and treatment diets are presented in Tables 5-7. In shrimp fed the control diet, saturated and monounsaturated

Table 5 Fatty acid content (mg gFatty acid

140 16:O

Experiment

’ dry matter) in hepatopancreas

of P. monodon fed the experimental

1

Experiment

diets

2

Control

Ref.

LI

NC

AR

EP

DH

Control

Ref.

ES

DS

S

0.4b 11.6b 2.3b 2.2c 14.0a 8.6b 1.oc 0.4c 0.9e 0.9d nd I.lf 14.2b 16.7b

0.8a t7.9a 3.2a 3.0a 13.la 9.4a 1.4b 2.4a 1.5de 10.3a nd 6.0a 21.7a 19.la

0.4b 9.ld 0.9e 1.7d 6.2d 2.9f l.oC 0.3c 5Sbc 2.9c nd 3.6c ll.W 7.4f

0.4b 10.6bc 1.4d 2.lc 7.4d 3.8de 0.4d 0.4c 5.2~ 3.lc nd 2.6d 12.6c 9.lde

OSb 10.5~ 1.9c 2.5b ll.lb 4.5~ 1.8a 0.7b 2.ld 5.2b 0.3 5.4a 13.4bc 13.2c

0.2~ 8.4de 0.4f 1.7d 7.4d 4.lcd 0.9c 0.3c 7.3a 1.2d nd 4.6b 10.4d 7.9ef

0.4b 7.8e l.lde 2.0~ 8.7~ 3.5ef 1.oc 0.4c 6.0b 3.8~ 0.2 1.9e 10% 10.2d

0.4b 12.8b 2.4b 2.0bc 16.Oa 9.6a 1.2a 0.4b 0.9c 1.2d nd 1.2d 15.2b 18.7a

0.6a 16.6a 3.la 2.6a 12Sb 9.0a l.Ob 2.0a 1.6b 8.9a nd 5.8a 19.8a 17.3a

0.2~ 6.6~ 0.5~ 1.8cd 4.0~ 2.2b 0.4c 0.4b 2.5a 5.3b nd 2.8~ 8.61~ 5.0b

0.2c 5.5~ 0.6~ 11% 5.oc 2.7b 0.3~ 0.3b 0.7~ 3.8~ nd 5.6a 7.2~ 5.8b

0.2c 5.8~ 0.6c 2.lb 4.3c 2.7b 0.3~ 0.4b 1.6b 4.5bc nd S.Ob 8.Oc 4.9b

17.6a

7.6c

6.1~

11.5b

6.61~

6.6~

3.6d

15.6a

8.4c

9.7b

9.3bc

10.9a

8.5b

8.5b

6.6c

10.9a

9.5b

10.6a

10.6a

4.7b

3.4c

4.3bc

69.2a

34.7~

36.2~

45.3b

36.Oc

36.4c

47.9b

63.3a

28.9c

26.5~ 26.2~

16:ln-7 18:0 l&In-9 18:2n-6 18:3n -3 20:ln-9 20:4n - 6 20:5n-3 220 22:6n-3 Sat a Monounsatnrates ’ n-3 3.ld PUFA ’ n-6 9Sb PUFA ’ 43.41, Total

’ Saturated: 14:0, 16:0, 18:O. b Monounsaturates: 16: 1n - 7, 18: 1 n - 9, 20: 1 n - 9. ‘n-3 polyunsaturated: 18:3n-3, 20:5n-3, 22:6n-3. d n - 6 polyunsaturated: 18:2n - 6, 20:4n - 6. Data are mean values of five replicates. Means within same rows with the same letters within each experiment are not significantly (P > 0.05) different. nd, not detected; tr, trace ( < 0.05 mg g-l).

284

Z.O. Merican, K.F. Shim/Aquaculture

Table 6 Fatty acid content (mg gFatty acid

Experiment Control

140 0.1 16:0 3.9b 16:ln-7 0.3a 18:O 0.7b 18:In-9 3Sab 18:2n-6 1.9a 18:3n -3 0.2b 20:ln-9 O.lb 20:4n - 6 0.4de 20:5n-3 0.8e 220 nd 22:6n - 3 1.oc Sat a 4.5b Monoun3.8b saturates b 1.9d n-3 PUFA’ n-6 2.2bcd PUFA ’ Total 12.5~

147 (1996) 275-291

’ dry matter) in polar lipids in tail muscle of P. monodon fed the experimental

1

Experiment

Ref.

Ll

NC

AR

0.1 3.5b 0.3a 1.Oa 2.Od 1.7a 0.2b 0.2ab 0.3e 2.3a nd 1.6b 4.7b 2.5e

0.1 0.1 0.1 4.Ob 4.0b 4.0b 0.2b 0.2b 0.2b 0.7b 0.7b 0.7b 2.6cd 2.8~ 2.9bc 1.2b 1.3b 1.4b 0.2b 0.1~ 0.2b O.lb O.lb 0.4a 1% 1.2~ 0.6d l.ld 1.3~ 1.6b nd nd nd 1.4b 1Sb 2.0a 4.7b 4.7b 4.8b 2.9cde 3.lcd 3.3~

diets

2

EP

DH

Control

Ref.

ES

0.1 4.7~1 0.2c 0% 3.6a 1.9a 0.4a 0.2ab 2.2a 0.8e nd 2.3a 5.5a 4.5a

0.1 3.7b 0.2c 0.7b 2.4cd 1.2b 0.2b 0.2ab 1.5b 1.4c 0.1 1.3b 4.5b 2.7de

0.1 4.2ab 0.3a 0.8ab 2.8b 2.la 0.2a 0. lb 0.4c I.ld nd l.ld 5.la 3.2ab

0.1 3.8b 0.3a 0.9ab 2.Oc 1Sb 0.2a 0.4a 0.4c 2.7a nd 2.2b 4.9a 2.7b

nd 0.1 4.4ab 4.8a 0.2b O.lc 0.8b 0.8ab 3.5a 2.6b 1.2b 1.5b O.lb O.lb O.lb O.lb 0.9a 0.4c 1.6~ 2.6a 0.1 nd 1.61~ 3.3a 5.7a 5.2a 3.8a 2.8b

DS

S 0.1 4.2ab 0.2b 0.9a 3.0ab 1.3b O.lb O.lb 0.8b 2.2b 0.1 2.lb 5.la 3.4ab

4.la

2.7~

2.8~

3.8a

3.3b

2.8~

2.3~

5.0a

4.2a

5.0a

4.4ab

1.9d

2.3bc

2.5b

1.9cd

4.0a

2.6b

2.6a

1.9b

2.Ob

1.9b

2.lb

13.2bc

12.6bc

13.4bc 13.7b

17.3a

12.6bc 12.1~

14.5ab

14.3b

16Sa

15.Oab

a Saturated: 140, 16:0, 18:O. b Monounsaturates: 16: 1 n - 7, 18: 1n - 9, 20: 1n - 9. ’ n-3 polyunsaturated: 18:3n-3, 20:5n-3, 22:6n-3. d n - 6 polyunsaturated: 18:2n - 6, 20:4n - 6. Data are mean values of five replicates. Means within same rows with the same letters within each experiment are not significantly (P > 0.05) different. nd, not detected; tr, trace f < 0.05 mg g- ’ ).

fatty acids amounted to 52.4-71% in tissues; similar to that in preconditioned shrimp (53.8-69.4%). With the supplementation of EFAs, tissue content of both 16:0 and 18: 1n - 9 reduced significantly (P < 0.05) in the hepatopancreas. Minor changes occurred in tail muscle lipids. Tissue levels of the n - 3 fatty acids deleted from the lipid mixture decreased significantly (P < 0.05) in all dietary treatments. However, the content of 18:3n - 3 in shrimp fed Diet NC was less than levels of 18:2n - 6, 20:4n - 6, 20:5n - 3 and 22:6n - 3 in shrimp fed Diets LI, AR, EP and DH, respectively. Irrespective of diet, shrimp also retained consistently low levels of 18:3n - 3 as compared with other n - 3 PUFAs. In shrimp fed Diet AR, containing all the n - 3 EFAs, 1.8 mg g-’ 18:3n - 3 was retained as compared with 5.2 mg g-’ 20:5n - 3 and 5.4 mg g-’ 22:6n - 3. A similar pattern was found in polar and neutral lipids of tail muscle. Although shrimp were fed diets without 22:6n - 3, levels of this fatty acid retained in tissues were relatively higher than that present in control shrimp. In contrast, 20:Sn - 3 retained in shrimp fed diets without 20:5n - 3 (Diet EP) was not significantly

Z.O. Merican, K.F. Shim/Aquaculture Table 7 Fatty acid content (mg gdiets Fatty acid

Experiment Control

14:o tr 0.6a 16:O 16:ln-7 0.1 0.2b 18:O 18:ln-9 I .Oa 18:2n-6 0.6a 18:3n-3 0.1 20:1n-9 tr 20:4n-6 O.le 20:5n - 3 0.3d nd 220 226n - 3 0.3d 0.9a Sat a Monounl.la saturates b n-3 OS% PUFA ’ 0.7d n-6 PUFA d 3.3c Total

147 (1996) 275-291

285

’ dry matter) in neutral lipid of taif muscle of P. monodon fed the experimental

1

Experiment

2

Ref.

LI

NC

AR

EP

DH

Control

Ref.

ES

nd 0.5b 0.1 O.ld 0.3e 0.3b 0.1 tr O.le 1.Oa nd 0.4cd 0.6b 0.4d

nd 0.4b 0.1 0.2c 0.7c 0.3b 0.1 tr 0.4c 0.5c nd 0.5bc 0.6b 0.8b

tr 0.4b 0.1 0.2b 0.7~ 0.3b 0.1 tr 0.5b 0.5~ nd 0.5b 0.6b 0.8b

tr 0.6a 0.1 0.2b 0.9bc 0.6a 0.1 0.1 0.3d 0.8b nd 0.7a 0.9a l.Oa

tr 0.4b tr O.ld O&I 0.3b 0.1 tr 0.5b 0.2d nd 0.4cd 0.6b 0.6c

tr 0.6a 0.1 0.4a 0.9ab 0.6a 0.2a 0.1 0.9a 0.8b nd 0.5bc l.Oa l.la

tr 0.5a 0.1 0.3a 0.6a OSa 0.2a tr O.lb 0.3d nd 0.3~ 0.8a 0.7a

tr 0.4b 0.1 O.lb 0.3bc 0.3b O.lb 0.1 O.lb 0.9a nd 0.6a 0.5b 0.5b

nd nd 0.2d 0.3~ tr tr 0.1~ O.lbc 0.3~ 0.4b O.ld 0.2~ tr tr nd nd 0.2a O.lb 0.5b 0.4bc nd nd 0.2d 0.5b 0.3c 0.4c 0.3d 0.4bc

1.5ab

0.7~

0.7~

1.6a

0.7c

1.4b

0.7c

1.6a

0.8~

0.9b

0.7c

0.4e

1.Ob

l.lb

0.8~

0.8~

1.5a

0.7a

0.5b

0.3c

0.3c

0.3c

2.9cd

3.Oc

3.3~

4.3b

2.6d

4.85a

2.9b

3.la

1.6d

2.oc

l&l

DS

S nd 0.2d tr O.lb 0.3bc O.ld O.lb nd O.lb 0.4bc nd 0.3c 0.3d 0.3cd

a Saturated: 140, 16:0, 18:0. b Monounsaturates: 16: I n - 7, 18: 1n - 9, 20: 1n - 9. ’ n - 3 polyunsaturated: 18:3n - 3, 20:5n - 3, 22:6n - 3. d n - 6 polyunsaturated: 18:2n - 6, 20:4n - 6. Data are mean values of five replicates. Means within same rows with the same letters within each experiment are not significantly (P > 0.05) different. nd, not detected; tr, trace ( < 0.05 mg g-l).

different (P > 0.05) from that in control shrimp. Similarly, in Experiment 2, elevated levels of 20:5n - 3 and 22:6n - 3 were present in the hepatopancreas and polar lipids of shrimp fed diets not supplemented with these n - 3 HUFAs. In the absence of 18:3n - 3 (Diet NC), shrimp apparently metabolised 22:6n - 3 as components of cellular membranes (D’Abramo and Sheen, 1993). This was indicated by the lower level of 22:6n - 3 present in the hepatopancreas. In contrast, 20:5n - 3 was utilised by being incorporated at higher levels in the lipid. A comparison of the tissue content of 18:2n - 6 in shrimp fed the control diet and that of shrimp fed diets supplemented with EFAs, showed that the latter group retained reduced levels of this fatty acid in the hepatopancreas. High levels of 20:4n - 6 were found in polar lipids of shrimp fed Diet EP and in neutral lipids of shrimp fed Diet DH. In contrast, significantly (P < 0.05) higher tissue levels of 20:4n - 6 occurred in shrimp fed diets with 1% 20:5n - 3, whereas levels decreased with the supplementation of 22:6n - 3.

286

Z.O. Merican, K.F. Shim /Aquaculture

147 (1996) 275-291

4. Discussion 4.1. Preconditioning Preconditioning of shrimp was required to enable a clearer observation of the patterns of use and possible essentiality of fatty acids. This period was also limited to 7 days to avoid stress and to reduce size variability. During this period, endogenous n - 3 PUFAs were depleted rapidly, because of the low levels of metabolic reserves (Cuzon et al., 1980). Major changes in the hepatopancreas were consistent with its role as a storage organ. The preferential utilisation of 140 and 16:0, rather than 18: 1 n - 9, in P. monodon was similarly reported by Sargent et al. (1989) in rainbow trout where dietary saturates were utilised rather than monounsaturates. In comparison, declines of > 75% in the proportions of all detectable n - 3 PUFA in whole body tissues of juveniles were obtained after 60 days of conditioning postlarvae Mucrobruchium rosenbergii with a EFA deficient diet (D’Abramo and Sheen, 1993). 4.2. Experimental

design

With these deletion experiments, it was possible to study the effects of a specific EFA in the presence of other EFAs. A negative growth response would thus indicate a requirement for the deleted fatty acid. It was also possible to identify, from tissue composition, the associative effects of other EFAs in the biosynthesis and bioconversion capabilities of the shrimp. However, since in penaeids, lecithin is required for normal growth and survival (Hertrampf, 1991), the basal mixture contained a mean level of 0.4% of 18:2n - 6 in the dry diet. With this limitation, all diets contained 18:2n - 6 and the effects of Diet LI must be regarded as that with reduced levels of 18:2n - 6 rather than from an absence of this fatty acid. The basal diet must be nutritionally complete so that growth and survival can be correlated to dietary lipid supplements. Thus in all feeding trials, a control diet which was not EFA supplemented confirmed the nutritional quality of the basal diet and the good growth performance of shrimp fed the reference diet verified that other variables, such as quality of juvenile shrimp and experimental conditions, were adequate. Due to the consistent growth response of shrimp fed the reference diet, results between trials were comparable and confirmed that the above conditions were similar for all trials. In addition, feeding shrimp with cod liver oil, a natural lipid source, provided baseline data on the fatty acid composition’ of tissues for comparison with shrimp fed purified fatty acids. In comparison with shrimp fed a commercial diet, growth of shrimp fed the reference diet was 96%. Using the level of acceptability of at least 80 to 85% of growth achieved with a practical diet (Cuzon et al., 1994), this purified diet would be acceptable for penaeid nutrition studies. 4.3. Growth and surviual Among the n - 3 PUFAs, a requirement for pre-formed 18:3n - 3 and 22:6n - 3, in diets for P. monodon, was demonstrated with these deletion experiments. The absence

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147 (19961275-291

287

of either fatty acid in the diet significantly (P < 0.05) depressed growth. However when 22:6n - 3 was supplemented at 1% as the sole HUFA in Experiment 2, weight gain was high. In homeothermic animals, Holman (1986) suggested that as the dietary intake of n - 6 fatty acids was increased, n - 6 PUFAs increased in tissues and this suppressed the utilisation of n - 3 PUFAs. Thus, the differences in the ratio of n - 6/n - 3 PUFAs in tissues of shrimp fed the different dietary regimes in the two experiments may explain the varying growth responses. Eicosapentaenoic acid was not a limiting fatty acid for growth of P. monodon. With other penaeids, several authors (Guary et al., 1976; Kanazawa et al., 1977, 1979~; Deshimaru et al., 1979; Read, 1981) have attributed the good performance of shrimp fed marine oils specifically to the action of 20:5n - 3 and 22:6n - 3. However, by using oils, it was not possible to identify the action of the specific EFA since there was always a mixture of several different types of n - 3 PUFAs. The results of feeding diets containing single sources of either 22:6n - 3 or 20:5n - 3 were in contrast with the findings of Kanazawa et al. (1978, 1979a) where growth of P. juponicus was better with 1% 20:5n - 3. In this study, an additive effect of 22:6n - 3 to 20:5n - 3 occurred in shrimp fed a mixture of these fatty acids. In comparison, the growth achieved by M. rosenbergii fed for 90 days with 0.15% C 2 20: n - 3 which comprised 30% 20:5n - 3 and 46.4% 22:6n - 3 was higher than that with 0.15% pure 22:6n - 3 (D’Abramo and Sheen, 1993). This growth response of shrimp fed diets with 1% 20:5n - 3 was in contrast to the positive growth response of shrimp fed the diet without 20:5n - 3 in Experiment 1. This may be explained by the use of this fatty acid as a n - 3 PUFA source, in the absence of either 18:3n - 3 or 22:6n - 3. With 1% 20:5n - 3 as the dietary EFA, growth was only 75% of that achieved with the same amount of 22:6n - 3 and thus a 0.75 relative efficacy of 20:5n - 3 to 22:6n - 3 was estimated. In red sea bream, the EFA efficiency of 22:6n - 3 was twice that of 20:5n - 3 (Takeuchi et al., 1990). A more conclusive value for P. monodon should be determined using diets with varying proportions of these fatty acids. Arachidonic acid was not a limiting fatty acid for P. monodon in the presence of sufficient n - 3 PUFAs. Within the current experimental design, it was difficult to determine whether 18:2n - 6 was equally important in P. monodon. In M. rosenbergii, growth was enhanced when fed a combination of 22:6n - 3 and 20:4n - 6 or 18:2n - 6 (D’Abramo and Sheen, 1993). This may explain the good growth of P. monodon fed Diets EP (with 22:6n - 3, 18:3n - 3 and complete n - 6 PUFAs) and AR (with 18:2n - 6 and complete n - 3 PUFAs). 4.4. Fatty acid composition Elevated levels of 18: 1 n - 9 in shrimp fed control diets was also a characteristic of EFA deficiency in marine fish (Takeuchi et al., 1990). No 20:2n - 9 and 20:3n - 9, the normal PUFAs to accumulate in EFA deficiency studies in fish (Tocher and Mackinlay, 19901, were detected in shrimp fed the control diets. Although this may be indicative of low A6 and A5 desaturase and elongase activities, a competitive inhibition between n - 9 and n - 6 acids in the control diets may have affected their metabolism (Holman, 1986). The presence of 0.4% 18:2n - 6 from lecithin might be sufficient to inhibit the

288

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147 (1996) 275-291

conversion of 18: 1 n - 9 to the C20 PUFA. The addition of n - 3 and n - 6 PUFAs resulted in reduced levels of saturates and monounsaturates. Depressed levels of 18: 1n - 9 in shrimp fed EFA diets may be explained by either the blocking of A9 desaturase enzyme by dietary 2 C20 PUFA or the use of abundant levels of these fatty acids as an energy source (Castell, 1982). The composition of n - 3 fatty acids present in tail muscle of shrimp fed treatment diets showed that P. monodon responded to the missing fatty acid. Significantly (P < 0.05) lower levels of 18:3n - 3 in tail muscle in shrimp fed diets without this fatty acid indicated that the synthesis of 18:3n - 3 apparently did not occur, similar to P. sefiferus (Bottino et al., 1980) and P. juponicus (Kanazawa et al., 1979a) as well as other decapods (M. rosenbergii, D’Abramo and Sheen, 1993). This observation supported the work of previous authors on the role of 18:3n - 3 as an essential fatty acid in other penaeids (Kanazawa et al., 1979a; Bottino et al., 1980; Read, 1981; Xu et al., 1994). It was also postulated that 18:3n - 3 was utilised for energy rather than bioconversion because no significant increase in the intermediate fatty acid 20:5n - 3 levels were found in shrimp fed the diet lacking in the latter fatty acid. However, a significant decrease in 22:6n - 3 in the hepatopancreas lipid of shrimp fed the diet with decreased 18:3n - 3, but with 20:5n - 3 and 22:6n - 3 present, suggested that there is chain elongation and desaturation of 18:3n - 3 to 22:6n - 3. It was difficult to ascertain this because no 20:3n - 3, by products of elongation, was detected. Elevated tissue levels of 22:6n - 3 in shrimp fed the diet without this fatty acid or containing only 1% 20:5n - 3 as EFA implied some chain elongation and desaturation of 20:5n - 3 to 22:6n - 3. Similarly, in shrimp fed the diet without 22:6n - 3, 20:5n - 3 should be able to substitute for 22:6n - 3 as EFA. This was not apparent probably because of the lower EFA efficiency of 20:5n - 3 relative to 22:6n - 3 and that percentage incorporation of 20:5n - 3 to 22:6n - 3 was insufficient to meet the requirements of the shrimp for growth. In marine fish, Watanabe et al. (1989) suggested that 22:6n - 3 was selectively utilised as energy for tissue development or converted to physically important derivatives. Thus whether 20:5n - 3 could substitute for 22:6n - 3 will depend on the physiological functions of these HUFAs in shrimp. This is still unknown in shrimp. In P. japonicus, no bioconversion of 20:5n - 3 to 22:6n - 3 occurred, particularly if the former has a higher value as EFA (Kanazawa et al., 1978; Teshima et al., 1992). However, in P. monodon, this may be plausible, since 22:6n - 3 was more effective as EFA than 20:5n - 3. However, in marine fish, where the efficacy of 22:6n - 3 was higher than 20:5n - 3, 20:5n - 3 was converted to 22:5n - 3 but not to 22:6n - 3. Docosahexaenoic acid was not retroconverted to 20:5n - 3 (Watanabe et al., 1989; Takeuchi et al., 1990, 1992), whereas elevated levels of 20:5n - 3 in shrimp fed diets with 1% 22:6n - 3 implied retroconversion from 22:6n - 3 in the absence of dietary 18:3n - 3. This has not been reported in penaeids, but M. rosenbergii displayed a synthesis of 20:5n - 3 from 22:6n - 3. It was postulated that this pathway may exist for the crustacean under particular dietary conditions (D’ Abram0 and Sheen, 1993). This retroconversion may be indicative of some requirement for 20:5n - 3 for metabolic functions. From tissue fatty acid composition, it was also implied that there was utilisation of 22:6n - 3 over 20:5n - 3, in the absence of 18:3n - 3 and that of 18:3n - 3 over 22:6n - 3 when 20:5n - 3 was not available in the diet.

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147 (1996) 275-291

289

There was no apparent synthesis of 18:2n - 6 in shrimp fed diets without additional 18:2n - 6. Elevated levels of 20:4n - 6 in shrimp fed diets without this fatty acid indicated a bioconversion from 18:2n - 6, evident by the presence of 20:2n - 6 and a selective conservation in tissues. A low degree of conversion was also reported by Lilly and Bottino (1981) by assaying the level of enzyme activity in hepatopancreas microsomes of P. setzferus. Increased levels of 20:4n - 6 in shrimp with a dietary supply were similar to the findings of D’Abramo and Sheen (1993) with M. rosenbergii. Elevated levels of 20:4n - 6 in shrimp fed diets with 1% 20:5n - 3 and low levels in shrimp fed diets with 1% 22:6n - 3 may imply some physiological control of 20:4n - 6 incorporated into tissue (Caste11 and Boghen, 1979). Elevated levels of 20:5n - 3 in diets without 20:4n - 6 could not be explained but may be linked to the widespread and consistent occurrence of 20:4n - 6 and 20:5n - 3 in tissues of aquatic insects (Hanson et al., 1985).

5. Conclusion With this experimental design, it was possible to conclude that pre-formed 18:3n - 3 and 22:6n - 3 were required by P. mono&m for growth, notwithstanding the composition of n - 3 and n - 6 PUFAs in the diet. Latent differences in requirements as compared with P. juponicus were evident. However, the essentiality of 18:2n - 6 was not fully established and adjustments to the experimental diets may be necessary. Whether these requirements differ at different levels of substrates should also be investigated. Further to this, an assessment of the nutrient requirement level of 18:3n - 3 and 22:6n - 3 should follow.

Acknowledgements We wish to thank Gold Coin Specialities Sdn Bhd. Malaysia for the use of tank facilities, Mr Chua Whye Leng for his technical support in the fatty acid analysis, Mr Isnin Jalil of the Gold Coin Farm and the staff of the Brackishwater Research Centre, Gelang Patah for their assistance. We also thank Dr Dean Akiyama and Mr Kenneth Chin for their advice, and the Director General, Fisheries Department Malaysia for granting study leave to Z.O. Merican to enable her to carry out this research. This research was conducted under grant #910405 from the National University of Singapore.

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Bottino, N.R., Gemtity, J., Lilly, M.L., Simmons, E. and Fmne, G., 1980. Seasonal and nutritional effects on the fatty acids of three species of shrimp, Penaeus setiferus, P. artecus and P. duorarum. Aquaculture, 19: 139-14s. Castell, J.D., 1982. Fatty acid metabolism in crustaceans. In: G.D. Pruder, C.J. Langdon and D.E. Conklin (Editors), Proceedings of the Second International Conference on Aquaculture Nutrition: Biochemical and Physiological Approaches to Shellfish Nutrition, Delaware. Special Publication No. 2, World Mariculture Society, pp. 124- 145. Castell, J.D. and Boghen, A., 1979. Fatty acid metabolism in juvenile lobsters (Homarus americanus) fed a diet low in methionine and histidine. Proc. World Maricult. Sot., 10: 720-727. Chen, H.-Y., 1995. Nutrition of Penaeus monodon. In: Abstracts of the Fifth International Working Group on Crustacean Nutrition Symposium, Kagoshima University, Kagoshima, Japan, 36 pp. Cuzon, G., Cahu, C., Aldrin, J.F., Messager, J.L., Stephan, G. and Mevel, M., 1980. Starvation effects on metabolism of Penaeus japonicus. hoc. World Maricult. Sot., 11: 410-423. Cuzon, G., Guillaume, J. and Cahu, C., 1994. Composition, preparation and utilization of feeds for Crustacea. Aquaculture, 124: 253-267. D’Abramo, L.R. and Sheen, S.-S., 1993. Polyunsaturated fatty acid nutrition in juvenile freshwater prawn Macrobrachium rosenbergii. Aquaculture, 115: 63-86. Deshimaru, O., Kuroki, K. and Yone, Y., 1979. The composition and level of dietary lipid appropriate for growth of prawn. Bull. Jpn. Sot. Sci. Fish., 45: 591-594. Folch, J., Lees, M. and Sloane Stanley, G.H., 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem., 226: 497-509. Guary, J.C., Kayama, M., Murakami, Y. and Ceccaldi, H.J., 1976. The effects of a fat-free diet and compounded diets supplemented with various oils on moult, growth and fatty acid composition of prawn, Penaeus japonicus Bate. Aquaculture, 7: 245-254. Hanson, B.J., Cummins, K.W., Cargill, A.S. and Lowry, R.R., 1985. Lipid content, fatty acid composition and the effect of diet on fats of aquatic insects. Comp. B&hem. Physiol., SOB: 257-276. Hertrampf, J.W., 1991. Feeding Aquatic Animals with Phospholipids I. Crustaceans. Publication No. 8, Lucas Meyer (GmbH and Co), KG, Hamburg, 31 pp. Holman, R.T., 1986. Nutritional and biochemical evidence of acyl interaction with respect to essential polyunsaturated fatty acids. Prog. Lipid. Res., 25: 29-39. Joseph, J.D. and A&man, R.G., 1992. Capillary column gas chromatography method for analysis of encapsulated fish oils and fish oil ethyl esters: collaborative study. J. AOAC Int., 75: 488-506. Juaneda, P. and Rocquelin, G., 1985. Rapid and convenient separation of phospholipids and non phospholipids from rat heart using silica cartridges. Lipids, 20: 40-41. Kanazawa, A., Teshima, S. and Tokiwa, S., 1977. Nutritional requirements of prawn - VII Effects of dietary lipids on growth. Bull. Jpn. Sot. Sci. Fish., 43: 849-856. Kanazawa, A., Teshima, S., Endo, M. and Kayama, M., 1978. Effects of eicosapentaenoic acid on growth and fatty acid composition of the prawn, Penaeus japonicus. Mem. Fat. Fish., Kagoshima Univ., 27: 35-40. Kanazawa, A., Teshima, S. and Endo, M., 1979a. Requirements of prawn, Penaeus japonicus for essential fatty acids. Mem. Fat. Fish., Kagoshima Univ., 28: 27-33. Kanazawa, A., Teshima, S. and Ono, K., 1979b. Relationship between essential fatty acid requirement of aquatic animals and the capacity for bioconversion of linolenic acid to highly unsaturated fatty acids. Comp. Biochem. Physiol., 63B: 295-298. Kanazawa, A., Teshima, S., Tokiwa, S., Endo, M. and Abdul Razak, F.A., 1979~. Effects of short necked clam phospholipids on the growth of prawn. Bull. Jpn. Sot. Sci. Fish., 45: 961-965. Kayama, M., Hirata, M., Kanazawa, A., Tokiwa, S. and Saito, M., 1980. Essential fatty acids in the diet of prawn - III Lipid metabolism and fatty acid composition. Bull. Jpn. Sot. Sci. Fish., 46: 483-488. Lilly, M.L. and Bottino, N.B., 1981. Identification of arachidonic acid in Gulf of Mexico shrimp and degree of biosynthesis in Penaeus setiferus. Lipids, 16: 871-875. Read, G.H.L., 1981. The response of Penaeus indicus (Crustacea: Penaeidea) to purified and compounded diets of varying fatty acid composition. Aquaculture, 24: 245-256. Sargent, J.R., Henderson, R.J. and Tocher, D.R., 1989. The lipids. In: J.E. Halver (Editor), Fish Nutrition. Academic Press, New York, pp. 153-218.

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SAS, 1987. SAS/STAT Guide for Personal Computers Version 6.03 Edition. SAS Institute Inc., Gary, NC, 378 pp. Takeuchi, T., Toyota, M., Satoh, S. and Watanabe, T., 1990. Requirement of juvenile red sea bream Pagrus major for eicosapentaenoic and docosahexaenoic acids. Nippon Suisan Gaikkashi., 56: 1263- 1269. Takeuchi, T., Arakawa, T., Satoh, S. and Watanabe, T., 1992. Supplemental effect of phospholipid and requirement of eicosapentaenoic and docosahexaenoic acids of juvenile striped jack. Nippon Suisan Gaikkashi., 58: 707-713. Teshima, S., Kanazawa, A., Hitotsumatsu, K.-T., Kim, K.-S., Oshida, K. and Koshio, S., 1992. Tissue uptake and bioconversion of icosapentaenoic and phosphatidylcholine in prawns, Penaeus and Macrohrachium. Comp. Biochem. Physiol., 102B: 885-890. Tocher, D.R. and Ma&inlay, E.E., 1990. Incorporation and metabolism of (n - 3) and (n - 6) polyunsaturated fatty acids in phospholipid classes in cultured turbot (Scopthalmus maximus) cells. Fish Physiol. B&hem., 8: 251-260. Wang, W.C., 1988. Growth rate and fatty acid composition of lipids of grass prawn fed with diets containing squid visceral oil. Qual. Control, 2: 13-28. Watanabe, T., Arakawa, T., Takeuchi, T. and Satoh, S., 1989. Comparison between eicosapentaenoic and docosahexaenoic acids in terms of essential fatty acid efficiency in juvenile striped jack Pseudocaranx dentex. Nippon Suisan Gaikkashi, 55: 1989- 1995. Xu, X.L., Ji, W.J., Castell, J.D. and Dor, R.K., 1994. Essential fatty acid requirements of the Chinese prawn, (Penaeus chinensis). Aquaculture, 127: 29-40.